Bottom Line:
We found that both PACAP38 and PACAP6-38, a selective PAC1 receptor antagonist, did not affect Y79 cell viability at nanomolar concentrations, but when used at 1-5 μM potently reduced cell survival in a dose-dependent manner.PACAP27 and maxadilan, a high affinity agonist of PAC1 receptors, had negligible effects.The cytotoxic effect of PACAP38 was augmented by p38, MEK1/2, and JNK inhibitors, indicating that high concentrations of the peptide might decrease the activity of these kinases, leading to cell death.

ABSTRACTPituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide expression of which has been found in various tumors of the brain and peripheral organs. Despite numerous studies, the exact role the peptide plays in the development and progression of tumors is not fully understood. In the present study, we investigated the effect of PACAP on human retinoblastoma Y79 cell viability. We found that both PACAP38 and PACAP6-38, a selective PAC1 receptor antagonist, did not affect Y79 cell viability at nanomolar concentrations, but when used at 1-5 μM potently reduced cell survival in a dose-dependent manner. PACAP27 and maxadilan, a high affinity agonist of PAC1 receptors, had negligible effects. Two membrane-penetrating analogs of PACAP38 inactive at PAC1/VPAC receptors, [Disc(6)]PACAP38 and FITC-Ahx-PACAP11-38, also decreased viability of Y79 cells, albeit with lower potency than PACAP38. The cytotoxic effect of PACAP38 was augmented by p38, MEK1/2, and JNK inhibitors, indicating that high concentrations of the peptide might decrease the activity of these kinases, leading to cell death. It is suggested that the cytotoxic activity of PACAP38 and PACAP6-38 against human retinoblastoma Y79 cell line may result from their interaction with target sites other than PAC1 and VPAC receptors, but this is yet unknown.

Fig3: Effect of [Disc6]PACAP38 and FITC-Ahx-PACAP11-38 on viability of Y79 cells. Cells were incubated with the peptides for 24 h and cell viability was analyzed by MTT test. Data are presented as mean ± SEM of 6–12 values per group and expressed as a percentage of the respective control.***p < 0.001 vs. control

Mentions:
Y79 cells were challenged with various peptides and cell viability, and mitochondrial function was measured by MTT test. Twenty-four-hour incubation of the cells with nanomolar (0.1–100 nM) concentrations of PACAP38 had no effect on their viability (data not shown). When the peptide was used in higher concentrations (1–5 μM) it produced a dose-dependent decrease in cell viability, with a calculated IC50 = 1.7 μM (Fig. 1a). The cytotoxic effect of PACAP38 was not abolished by a PAC1 receptor antagonist, PACAP6-38. In fact, PACAP6-38 (1–5 μM) also produced a concentration-dependent reduction of Y79 cell viability with a similar potency and IC50 = 2.1 μM (Fig. 1b). The suppressive effects of both peptides on Y79 cell viability were additive (Fig. 2). On the contrary, incubation of Y79 cells with PACAP27 (0.1–5 μM) and maxadilan (1 and 2 μM), a high affinity selective agonist of PAC1 receptors, did not significantly affect their viability (data not shown). Two membrane-penetrating analogs of PACAP38 inactive at PAC1/VPAC receptors, [Disc6]PACAP38 and FITC-Ahx-PACAP11-38, decreased viability of Y79 cells, albeit with lower potency than PACAP38 (Fig. 3). Another cell penetrating peptide, FITC-Ahx-TAT48-60, which has no structure similarity to PACAP, and membrane non-penetrating C-terminal fragment of PACAP, FITC-Ahx-PACAP28-38, used at a 10 μM concentration were inactive (data not shown).Fig. 1

Fig3: Effect of [Disc6]PACAP38 and FITC-Ahx-PACAP11-38 on viability of Y79 cells. Cells were incubated with the peptides for 24 h and cell viability was analyzed by MTT test. Data are presented as mean ± SEM of 6–12 values per group and expressed as a percentage of the respective control.***p < 0.001 vs. control

Mentions:
Y79 cells were challenged with various peptides and cell viability, and mitochondrial function was measured by MTT test. Twenty-four-hour incubation of the cells with nanomolar (0.1–100 nM) concentrations of PACAP38 had no effect on their viability (data not shown). When the peptide was used in higher concentrations (1–5 μM) it produced a dose-dependent decrease in cell viability, with a calculated IC50 = 1.7 μM (Fig. 1a). The cytotoxic effect of PACAP38 was not abolished by a PAC1 receptor antagonist, PACAP6-38. In fact, PACAP6-38 (1–5 μM) also produced a concentration-dependent reduction of Y79 cell viability with a similar potency and IC50 = 2.1 μM (Fig. 1b). The suppressive effects of both peptides on Y79 cell viability were additive (Fig. 2). On the contrary, incubation of Y79 cells with PACAP27 (0.1–5 μM) and maxadilan (1 and 2 μM), a high affinity selective agonist of PAC1 receptors, did not significantly affect their viability (data not shown). Two membrane-penetrating analogs of PACAP38 inactive at PAC1/VPAC receptors, [Disc6]PACAP38 and FITC-Ahx-PACAP11-38, decreased viability of Y79 cells, albeit with lower potency than PACAP38 (Fig. 3). Another cell penetrating peptide, FITC-Ahx-TAT48-60, which has no structure similarity to PACAP, and membrane non-penetrating C-terminal fragment of PACAP, FITC-Ahx-PACAP28-38, used at a 10 μM concentration were inactive (data not shown).Fig. 1

Bottom Line:
We found that both PACAP38 and PACAP6-38, a selective PAC1 receptor antagonist, did not affect Y79 cell viability at nanomolar concentrations, but when used at 1-5 μM potently reduced cell survival in a dose-dependent manner.PACAP27 and maxadilan, a high affinity agonist of PAC1 receptors, had negligible effects.The cytotoxic effect of PACAP38 was augmented by p38, MEK1/2, and JNK inhibitors, indicating that high concentrations of the peptide might decrease the activity of these kinases, leading to cell death.

ABSTRACTPituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide expression of which has been found in various tumors of the brain and peripheral organs. Despite numerous studies, the exact role the peptide plays in the development and progression of tumors is not fully understood. In the present study, we investigated the effect of PACAP on human retinoblastoma Y79 cell viability. We found that both PACAP38 and PACAP6-38, a selective PAC1 receptor antagonist, did not affect Y79 cell viability at nanomolar concentrations, but when used at 1-5 μM potently reduced cell survival in a dose-dependent manner. PACAP27 and maxadilan, a high affinity agonist of PAC1 receptors, had negligible effects. Two membrane-penetrating analogs of PACAP38 inactive at PAC1/VPAC receptors, [Disc(6)]PACAP38 and FITC-Ahx-PACAP11-38, also decreased viability of Y79 cells, albeit with lower potency than PACAP38. The cytotoxic effect of PACAP38 was augmented by p38, MEK1/2, and JNK inhibitors, indicating that high concentrations of the peptide might decrease the activity of these kinases, leading to cell death. It is suggested that the cytotoxic activity of PACAP38 and PACAP6-38 against human retinoblastoma Y79 cell line may result from their interaction with target sites other than PAC1 and VPAC receptors, but this is yet unknown.